Mobile phase aqueous solvents

In reversed-phase thin-layer chromatography (RP-TLC), the choice of solvents for the mobile phase is carried out in a reversed order of strength, comparing with the classical TLC, which determines a reversed order of values of compounds. The reversed order of separation assumes that water is the main component of the mobile phase. Aqueous mixmres of some organic solvents (diethyl ether, methanol, acetone, acetonitrile, dioxane, i-propanol, etc.) are used with good results. 

Different types of buffers at the same ionic strength and wpH can have a significant impact on the dissolution of silica. The dissolution of silica is usually measured by the silicomolybdate colorimetric method [41]. When determining the bonded-phase stability using different run buffers (effect of buffer counteranion or countercation), the same H must be used. The H values (pH of the mobile phase aqueous -i- organic) may be different from the aqueous portion of the mobile phase and may obscure if the dissolution of the silica is directly related to the type of anion/cation and/or the pH. Generally, with the addition of organic solvents the pH of the mobile phase decreases for basic buffers and increases for acidic buffers (see Section 4.5 for more details). 

The introduction of the chemically bonded apolar reversed phase materials as stationary phases is responsible for the common use of HPLC in routine laboratory analysis, because the sample can be applied directly from the aqueous solution into the HPLC-system. For the mobile phase, polar solvents such as water or aqueous buffer solutions or gradient mixes of aqueous solvents with methanol or acetonitrile are used mainly. 

Effect of acetonitrile in HPLC When the recovery of small molecules analyzed in serum is calculated based on the peak height of aqueous standards also treated by acetonitrile similar to serum, a high recovery is obtained. However, when the calculation is based on aqueous standards diluted in the mobile phase (pump solvent) a low recovery is obtained. In analyses of drugs in serum, acetonitrile in the sample decreases the peak height and limits the amount of sample to be injected on the column due to the formation of a short gradient leading to a non-symmetrical peak shape with a decrease in the plate number. Alternative deproteinizing mixtures for acetonitrile in HPLC have been proposed. 

Capillary electrochromatography has experienced rapid progress during the last decade, expanding from 17 publications in 1994 to 191 in 2007. This has also led to several books and reviews [93-104] and analytical instrumentation is readily commercially available [105]. The developments in CEC include research on optimum stationary phases (polymer or silica based, adsorbed or imprinted, etc.), mobile phases (aqueous electrolytes with/without admixture of organic solvents or pseudophases) and apparatus design (open-tubular, packed or monolithic capillaries) up to lab-on-a-chip devices for pTAS [107]. 

In GPC, the solvent in which the standards and sample are dissolved should be identical to the mobile-phase solvent in which the analysis will be performed. In most cases filtration is the only step needed to prepare the mobile phase. Organic solvents should be vacuum filtered through a 0.45-pm fluorocarbon filter, while acetate-type filters are used with aqueous mobile phases. In some cases mobile-phase additives are required. When polar solvents such as N,N-dimethyformamide, N,N-dimethylacetamide, and -methyl pyrrolidone are used to analyze polar polymers such as poly-... 

Solvent triangle for optimizing reverse-phase HPLC separations. Binary and ternary mixtures contain equal volumes of each of the aqueous mobile phases making up the vertices of the triangle. 

This experiment focuses on developing an HPLG separation capable of distinguishing acetylsalicylic acid, paracetamol, salicylamide, caffeine, and phenacetin. A Gjg column and UV detection are used to obtain chromatograms. Solvent parameters used to optimize the separation include the pH of the buffered aqueous mobile phase, the %v/v methanol added to the aqueous mobile phase, and the use of tetrabutylammonium phosphate as an ion-pairing reagent. 

In reeent years, tire use of elevated temperatures has been reeognised as a potential variable in method development. Witlr inereased temperature, aqueous-organie mobile phases separations ean improve, viseosity deereases and diffusion inereases so baek pressures are redueed. At higher temperatures (usually with superheated water > 100 °C under modest pressures) water alone ean be used as the mobile phase and eair provide unique separation opportunities. The absenee of an organie solvent enables the use in HPLC of alternative deteetors sueh as FID or on-line LC-NMR using deuterium oxide as the eluent. 

Silica gel, per se, is not so frequently used in LC as the reversed phases or the bonded phases, because silica separates substances largely by polar interactions with the silanol groups on the silica surface. In contrast, the reversed and bonded phases separate material largely by interactions with the dispersive components of the solute. As the dispersive character of substances, in general, vary more subtly than does their polar character, the reversed and bonded phases are usually preferred. In addition, silica has a significant solubility in many solvents, particularly aqueous solvents and, thus, silica columns can be less stable than those packed with bonded phases. The analytical procedure can be a little more complex and costly with silica gel columns as, in general, a wider variety of more expensive solvents are required. Reversed and bonded phases utilize blended solvents such as hexane/ethanol, methanol/water or acetonitrile/water mixtures as the mobile phase and, consequently, are considerably more economical. Nevertheless, silica gel has certain areas of application for which it is particularly useful and is very effective for separating polarizable substances such as the polynuclear aromatic hydrocarbons and substances... 

Zorbax PSM packings are produced in three forms unmodified, trimethyl-silane modified, and diol modified. Modified Zorbax PSM packings are produced by chemically bonding a layer on the silica surface through siloxane bonds (Table 3.1). Silanized Zorbax PSM packings suppress adsorption effects and are the preferred choice when the mobile phase contains organic solvents. Unsilanized and diol modified Zorbax PSM packings should be used when the mobile phase consists of aqueous solvents. 

For the size exclusion chromatography of proteins on silica-hased diol packings, it is generally recommended to use fully aqueous mobile phases with a salt concentration between 0.1 and 0.3 M. In general, a phosphate buffer around pH 7 is used as the mobile phase. Under these circumstances, the tertiary structure of most proteins is preserved without difficulty and the interaction of proteins with each other is minimized. However, other inorganic buffers or combinations of buffers with organic solvents can be used without difficulties for special applications. 

Even with mobile-phase modifiers, however, certain polymer types cannot be run due to their lack of solubility in organic solvents. In order to run aqueous or mixed aqueous/organic mobile phases, Jordi Associates has developed several polar-bonded phase versions of the PDVB gels as discussed earlier. Figures 13.60 thru 13.99 detail examples of some polar and ionic polymers that we have been able to run SEC analysis of using the newer bonded PDVB resins. 

Traditionally, LC and GC are used as separate steps in the sample analysis sequence, with collection in between, and then followed by transfer. A major limitation of off-line LC-GC is that only a small aliquot of the LC fraction is injected into the GC p. (e.g. 1 - 2 p.1 from 1 ml). Therefore, increasing attention is now given to the on-line combination of LC and GC. This involves the transfer of large volumes of eluent into capillary GC. In order to achieve this, the so-called on-column interface (retention gap) or a programmed temperature vaporizor (PTV) in front of the GC column are used. Nearly all on-line LC-GC applications involve normal-phase (NP) LC, because the introduction of relatively large volumes of apolar, relatively volatile mobile phases into the GC unit is easier than for aqueous solvents. On-line LC-GC does not only increase the sensitivity but also saves time and improves precision. 

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